HIV diagnostic methods

ABSTRACT

A peptide has an amino acid sequence having more than 80% homology with the amino acid sequence listed as SEQ ID NO:4. A nucleic acid molecule has more than 80% homology with one of the nucleic acid sequences listed as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3. Ligands, anti-ligands, cells vectors relating to the peptide and/or nucleic acid molecule are also used.

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/938,703, filed Aug. 24, 2001, which is a divisional of U.S.application Ser. No. 09/626,939, filed Jul. 27, 2000, which is adivisional of U.S. application Ser. No. 08/833,752, filed Apr. 9, 1997,which is a continuation of U.S. application Ser. No. 08/810,028, filedMar. 4, 1997, which claims the benefit of EP 96870021.1, filed Mar. 1,1996 and EP 96870102.9, filed Aug. 6, 1996. The entire teachings of theabove applications are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The present invention concerns new peptides and the nucleic acidmolecules encoding said peptides, the vector comprising said nucleicacid molecules, the cells transformed by said vector, inhibitorsdirected against said peptides or said nucleic acid molecules, apharmaceutical composition and a diagnostic and/or dosage devicecomprising said products, and non human transgenic animals expressingthe peptides according to the invention or the nucleic acid moleculesencoding said peptides.

[0003] The invention further provides a method for determining ligandbinding, detecting expression, screening for drugs binding specificallyto said peptides and treatments involving the peptides or the nucleicacid molecules according to the invention.

TECHNOLOGICAL BACKGROUND OF THE ART

[0004] Chemotactic cytokines, or chemokines, are small signallingproteins that can be divided in two subfamilies (CC— and CXC-chemokines)depending on the relative position of the first two conserved cysteines.Interleukin 8 (IL-8) is the most studied of these proteins, but a largenumber of chemokines (Regulated on Activation Normal T-cell Expressedand Secreted (RANTES), Monocyte Chemoattractant Protein 1 (MCP-1),Monocyte Chemoattractant Protein 2 (MCP-2), Monocyte ChemoattractantProtein 3 (MCP-3), Growth-Related gene product α (GROα), Growth-Relatedgene product β (GRO)β, Growth-Related gene product γ (GROγ), MacrophageInflammatory Protein 1 α (MIP-1α) and β, etc.) has now been described[4]. Chemokines play fundamental roles in the physiology of acute andchronic inflammatory processes as well as in the pathologicaldysregulations of these processes, by attracting and simulating specificsubsets of leucocytes [32]. RANTES for example is a chemoattractant formonocytes, memory T-cells and eosinophils, and induces the release ofhistamine by basophils. MCP-1, released by smooth muscle cells inarteriosclerotic lesions, is considered as the factor (or one of thefactors) responsible for macrophage attraction and, therefore, for theprogressive aggravation of the lesions [4].

[0005] MIP-1α, MIP-1β and RANTES chemokines have recently been describedas major HIV-suppressive factors produced by CD8⁺ T-cells [9].CC-chemokines are also involved in the regulation of human myeloidprogenetor cell proliferation [6, 7].

[0006] Recent studies have demonstrated that the actions of CC— andCXC-chemokines are mediated by subfamilies of G protein-coupledreceptors. To date, despite the numerous functions attributed tochemokines and the increasing number of biologically active ligands,only six functional receptors have been identified in human. Tworeceptors for interleukin-8 (IL-8) have been described [20, 29]. One(IL-8RA) binds IL-8 specifically, while the other (IL-8RB) binds IL-8and other CXC-chemokines, like GRO. Among receptors bindingCC-chemokines, a receptor, designated CC-chemokine receptor 1 (CCR1),binds both RANTES and MIP-1α [31], and the CC-chemokine receptor 2(CCR2) binds MCP-1 and MCP-3 [8, 44, 15]. Two additional CC-chemokinereceptors were cloned recently: the CC-chemokine receptor 3 (CCR3) wasfound to be activated by RANTES, MIP-1α and MIP-1β [10]; theCC-chemokine receptor 4 (CCR4) responds to MIP-1, RANTES and MCP-1 [37].In addition to these six functional receptors, a number of orphanreceptors have been cloned from human and other species, that arestructurally related to either CC— or CXC-chemokine receptors. Theseinclude the human BLR1 [13], EBI1 [5], LCR1 [21], the mouse MIP-1 RL1and MIP-1 RL2 [17] and the bovine PPR1 [25]. Their respective ligand(s)and function(s) are unknown at present.

SUMMARY OF THE INVENTION

[0007] The present invention is related to a peptide having at least anamino acid sequence which presents more than 80%, advantageously morethan 90%, preferably more than 95%, homology with the amino acidsequence as represented in SEQ ID NO. 1.

[0008] Preferably, said peptide has also at least an amino acid sequencewhich presents more than 80%, advantageously more than 90%, preferablymore than 95%, homology with the amino acid sequence as represented inSEQ ID NO. 2.

[0009] According to another embodiment of the present invention, thepeptide has at least an amino acid sequence which presents more than80%, advantageously more than 90%, preferably more than 95%, homologywith the amino acid sequence as represented in SEQ ID NO. 3.

[0010] The present invention is also related to the amino acid sequenceof SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 or a portion thereof(represented in the FIG. 1).

[0011] A “portion of an amino acid sequence” means one or more aminoacid segments having the same or improved binding properties of thewhole peptide according to the invention. Said portion could be anepitope which is specifically binded by a ligand of the peptide whichcould be a known “natural ligand” of said peptide, an agonist or ananalog of said ligand, or an inhibitor capable of competitivelyinhibiting the binding of said ligand to the peptide (including theantagonists of said ligand to the peptide).

[0012] Specific examples of said portions of amino acid sequence andtheir preparation process are described in the publication of Rucker J.et al. (Cell, Vol. 87, pp. 437-446 (1996)) incorporated herein byreference.

[0013] According to the invention, said portion of the amino acidsequence of the peptide according to the invention comprises theN-terminus segment and the first extracellular loop of the peptide.

[0014] Therefore, according to the invention, the amino acid sequence asrepresented in SEQ ID NO. 1 is the common amino acid sequence of SEQ IDNO. 2 and of SEQ ID NO. 3 (see also FIG. 1). Therefore, a firstindustrial application of said amino acid sequence is the identificationof the homology between said amino acid sequence and the screening ofvarious mutants encoding a different amino acid sequence than the onepreviously described, and the identification of various types of patientwhich may present a predisposition or a resistance to the disordersdescribed in the following specification.

[0015] Preferably, the peptide according to the invention or a portionthereof is an active CC-chemokine receptor.

[0016] Advantageously, the CC-chemokine receptor according to theinvention is stimulated by the MIP-1β chemokine at a concentration lessor equal to 10 nm, and is advantageously also stimulated by the MIP-1αor RANTES chemokines. However, said chemokine receptor is not stimulatedby the MCP-1, MCP-2, MCP-3, IL-8 and GROα chemokines.

[0017] In addition, the peptide according to the invention or a portionthereof is also a receptor of HIV viruses or a portion of said HIVviruses.

[0018] It is meant by “HIV viruses”, HIV-1 or HIV-2 and all the variousstrains of HIV viruses which are involved in the development of AIDS. Itis meant by a “a portion of HIV viruses”, any epitope of said viruseswhich is able to interact specifically with said receptor. Among saidportions of viruses which may be involved in the interaction with thepeptide according to the invention, are peptides encoded by the ENV andGAG viruses genes.

[0019] Preferably, said portion of HTV viruses is the glycopeptidegp120/160 (membrane-bound gp160 or the free gp derived therefrom) or aportion thereof.

[0020] It is meant by a “portion of the glycopeptide gp120/160” anyepitope, preferably an immuno-dominant epitope, of said glycopeptidewhich may interact specifically with the peptide according to theinvention, such as for instance the V3 loop (third hypervariabledomain).

[0021] According to another embodiment of the present invention, thepeptide according to the invention is an inactive CC-chemokine receptor.An example of such inactive CC-chemokine receptor is encoded by theamino acid sequence as represented in SEQ ID NO. 2.

[0022] It is meant by an “inactive CC-chemokine receptor” a receptorwhich is not stimulated by any known CC-chemokine, especially theMIP-1β, MIP-1α or RANTES chemokines.

[0023] The peptide represented in SEQ ID NO. 3 according to theinvention is an 30 inactive receptor which is not a receptor of HIVviruses or of a portion of said HIV viruses, which means that saidinactive receptor does not allow the entry of said FHV viruses into acell which presents at its surface said inactive receptor.

[0024] Advantageously, the peptide according to the invention is a humanreceptor.

[0025] The present invention concerns also the nucleic acid moleculehaving more than 80%, preferably more than 90%, homology with one of thenucleic acid sequences of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3shown in the FIG. 1.

[0026] Preferably, said nucleic acid molecule has at least the nucleicacid sequence shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3 ofFIG. 1 or a portion thereof.

[0027] It is meant by a “portion of said nucleic acid molecule” anynucleic acid sequence of more than 15 nucleotides which could be used inorder to detect and/or reconstitute said nucleic acid molecule or itscomplementary strand. Such portion could be a probe or a primer whichcould be used in genetic amplification using the PCR, LCR, NASBA or CPRtechniques for instance.

[0028] The present invention concerns more specifically the nucleic acidmolecules encoding the peptide according to the invention. Said nucleicacid molecules are RNA or DNA molecules such as a cDNA molecule or agenomic DNA molecule.

[0029] The present invention is also related to a vector comprising thenucleic acid molecule according to the invention. Preferably, saidvector is adapted for expression in a cell and comprises the regulatoryelements necessary for expressing the amino acid molecule in said celloperatively linked to the nucleic acid sequence according to theinvention as to permit expression thereof.

[0030] Preferably, said cell is chosen among the group consisting ofbacterial cells, yeast cells, insect cells or mammalian cells. Thevector according to the invention is a plasmid, preferably a pcDNA3plasmid, or a virus, preferably a baculovirus, an adenovirus or asemliki forest virus.

[0031] The present invention concerns also the cell, preferably amammalian cell, such as a CHO-K1 or a HEK293 cell, transformed by thevector according to the invention. Advantageously, said cell is nonneuronal in origin and is chosen among the group consisting of CHO-K1,HEK293, BHK21, COS-7 cells.

[0032] The present invention also concerns the cell (preferably amammalian cell such as a CHO-K1 cell) transformed by the vectoraccording to the invention and by another vector encoding a proteinenhancing the functional response in said cell. Advantageously, saidprotein is the Gα15 or Gα16 (G protein, α subunit). Advantageously, saidcellis the cell CHO-K1-pEFIN hCCR5-1/16.

[0033] The present invention is also related to a nucleic acid probecomprising a nucleic acid molecule of at least 15 nucleotides capable ofspecifically hybridizing with a unique sequence included within thesequence of the nucleic acid molecule according to the invention. Saidnucleic acid probe may be a DNA or a RNA.

[0034] The invention concerns also an antisense oligonucleotide having asequence capable of specifically hybridizing to an MRNA moleculeencoding the peptide according to the invention so as to preventtranslation of said MIRNA molecule or an antisense oligonucleotidehaving a sequence capable of specifically hybridizing to the CDNAmolecule encoding the peptide according to the invention.

[0035] Said antisense oligonucleotide may comprise chemical analogs ofnucleotide or substances which inactivate MRNA, or be included in an RNAmolecule endowed with ribozyme activity.

[0036] Another aspect of the present invention concerns a ligand or ananti-ligand (preferably an antibody) other than known “natural ligands”,which are chosen among the group consisting of the MIP-1β, MIP-1α orRANTES chemokines, HIV viruses or a portion of said HIV viruses, whereinsaid ligand is capable of binding to the receptor according to theinvention and wherein said anti-ligand is capable of (preferablycompetitively) inhibiting the binding of said known “natural ligand” orthe ligand according to the invention to the peptide according to theinvention.

[0037] The exclusion in the above identified definition of knownchemokines, HIV viruses or a portion of said HIV viruses, does notinclude variants of said “natural” viruses or said “natural” portionwhich may be obtained for instance by genetic engineering and which maymimic the interaction of said viruses and portion of said viruses to thepeptide according to the invention.

[0038] Advantageously, said antibody is a monoclonal antibody which ispreferably directed to an epitope of the peptide according to theinvention and present on the surface of a cell expressing said peptide.

[0039] Preferably, said antibody is produced by the hybridome cellAchCCR5-SAB1A7.

[0040] The invention concerns also the pharmaceutical compositioncomprising either an effective amount of the peptide according to theinvention (in order to delude the HIV virus from the natural peptidepresent at the surface of a mammalian cell and stop the infection ofsaid mammalian cell by the HIV virus), or an effective amount of theabove identified described ligand and/or anti-ligand, or an effectiveamount of oligonucleotide according to the invention, effective todecrease the activity of said peptide by passing through a cell membraneand binding specifically with MRNA encoding the peptide according to theinvention in the cell so as to prevent it translation. Thepharmaceutical composition comprises also a pharmaceutically acceptablecarrier, preferably capable of passing through said cell membrane.

[0041] Preferably, in said pharmaceutical composition, theoligonucleotide is coupled to a substance, such as a ribozyme, whichinactivates MRNA encoding the peptide according to the invention.

[0042] Preferably, the pharmaceutically acceptable carrier comprises astructure which binds to a receptor on a cell capable of being taken upby cell after binding to the structure. The structure of thepharmaceutically acceptable carrier in said pharmaceutical compositionis capable of binding to a receptor which is specific for a selectedcell type.

[0043] The present invention concerns also a transgenic non human mammaloverexpressing (or expressing ectopically) the nucleic acid moleculeencoding the peptide according to the invention.

[0044] The present invention also concerns a transgenic non human mammalcomprising an homologous recombination knockout of the native peptideaccording to the invention.

[0045] According to a preferred embodiment of the invention, thetransgenic non human mammal whose genome comprises antisense nucleicacid complementary to the nucleic acid according to the invention is soplaced as to be transcripted into antisense MRNA which is complementaryto the MRNA encoding the peptide according to the invention and whichhybridizes to MRNA encoding said peptide, thereby reducing itstranslation. Preferably, the transgenic non human mammal according tothe invention comprises a nucleic acid molecule encoding the peptideaccording to the invention and comprises additionally an induciblepromoter or a tissue specific regulatory element.

[0046] Preferably, the transgenic non human mammal is a mouse.

[0047] The invention relates to a method for determining whether aligand can be specifically bound to the peptide according to theinvention, which comprises contacting a cell transfected with a vectorexpressing the nucleic acid molecule encoding said peptide with theligand under conditions permitting binding of ligand to such peptide anddetecting the presence of any such ligand bound specifically to saidpeptide, thereby determining whether the ligand binds specifically tosaid peptide.

[0048] The invention relates to a method for determining whether aligand can specifically bind to a peptide according to the invention,which comprises preparing a cell extract from cells transfected with avector expressing the nucleic acid molecule encoding said peptide,isolating a membrane fraction from the cell extract, contacting theligand with the membrane fraction under conditions permitting binding ofthe ligand to such peptide and detecting the presence of any ligandbound to said peptide, thereby determining whether the compound iscapable of specifically binding to said peptide. Preferably, said methodis used when the ligand is not previously known.

[0049] The invention relates to a method for determining whether aligand is an agonist of the peptide according to the invention, whichcomprises contacting a cell transfected with a vector expressing thenucleic acid molecule encoding said peptide with the ligand underconditions permitting the activation of a functional peptide responsefrom the cell and detecting by means of a bio-assay, such as amodification in a second messenger concentration (preferably calciumions or inositol phosphates such as IP₃) or a modification in thecellular metabolism (preferably determined by the acidification rate ofthe culture medium), an increase in the peptide activity, therebydetermining whether the ligand is a peptide agonist.

[0050] The invention relates to a method for determining whether aligand is an agonist of the peptide according to the invention, whichcomprises preparing a cell extract from cells transfected with a vectorexpressing the nucleic acid molecule encoding said peptide, isolating amembrane fraction from the cell extract, contacting the membranefraction with the ligand under conditions permitting the activation of afunctional peptide response and detecting by means of a bio-assay, suchas a modification in the production of a second messenger (preferablyinositol phosphates such as IP₃), an increase in the peptide activity,thereby determining whether the ligand is a peptide agonist.

[0051] The present invention relates to a method for determining whethera ligand is an antagonist of the peptide according to the invention,which comprises contacting a cell transfected with a vector expressingthe nucleic acid molecule encoding said peptide with the ligand in thepresence of a known peptide agonist, under conditions permitting theactivation of a functional peptide response and detecting by means of abio-assay, such as a modification in second messenger concentration(preferably calcium ions or inositol phosphates such as IP₃) or amodification in the cellular metabolism (preferably determined by theacidification rate of the culture medium), a decrease in the peptideactivity, thereby determining whether the ligand is a peptideantagonist.

[0052] The present invention relates to a method for determining whethera ligand is an antagonist of the peptide according to the invention,which comprises preparing a cell extract from cells transfected with anexpressing the nucleic acid molecule encoding said peptide, isolating amembrane fraction from the cells extract, contacting the membranefraction with the ligand in the presence of a known peptide agonist,under conditions permitting the activation of a functional peptideresponse and detecting by means of a bio-assay, such as a modificationin the production of a second messenger, a decrease in the peptideactivity, thereby determining whether the ligand is a peptideantagonist.

[0053] Preferably, the second messenger assay comprises measurement ofcalcium ions or inositol phosphates such as IP₃.

[0054] Preferably, the cell used in said method is a mammalian cell nonneuronal in origin, such as CHO-K1, HEK293, BHK21, COS-7 cells.

[0055] In said method, the ligand is not previously known.

[0056] The invention is also related to the ligand isolated and detectedby any of the preceding methods.

[0057] The present invention concerns also the pharmaceuticalcomposition which comprises an effective amount of an agonist or anantagonist of the peptide according to the invention, effective toreduce the activity of said peptide and a pharmaceutically acceptablecarrier.

[0058] It is meant by “an agonist or an antagonist of the peptideaccording to the invention”, all the agonists or antagonists of theknown “natural ligand” of the peptide as above described.

[0059] Therefore, the previously described methods may be used for thescreening of drugs to identify drugs which specifically bind to thepeptide according to the invention.

[0060] The invention is also related to the drugs isolated and detectedby any of these methods.

[0061] The present invention concerns also a pharmaceutical compositioncomprising said drugs and a pharmaceutically acceptable carrier.

[0062] The invention is also related to a method of detecting expressionof a peptide according to the invention by detecting the presence ofMRNA coding for a peptide, which comprises obtaining total RNA or totalMRNA from the cell and contacting the RNA or MRNA so obtained with thenucleic acid probe according to the invention under hybridizingconditions and detecting the presence of MRNA hybridized to the probe,thereby detecting the expression of the peptide by the cell.

[0063] Said hybridization conditions are stringent conditions.

[0064] The present invention concerns also the use of the pharmaceuticalcomposition according to the invention for the treatment and/orprevention of inflammatory diseases, including rheumatoid arthritis,glomerulonephritis, asthma, idiopathic pulmonary fibrosis and psoriasis,viral infections including Human Immunodeficiency Viruses 1 and 2 (HIV-1and 2), cancer including leukaemia, atherosclerosis and/or auto-immunedisorders.

[0065] The present invention concerns also a method for diagnosing apredisposition or a resistance to a disorder associated with theactivity of the peptide according to the invention and/or associatedwith infectious agents such as HIV viruses in a subject. Said methodcomprises

[0066] a) obtaining nucleic acid molecules encoding the peptideaccording to the invention from the cells of the subject;

[0067] b) possibly performing a restriction digest of said nucleic acidmolecules with a panel of restriction enzymes;

[0068] c) possibly electrophoretically separating the resulting nucleicacid fragments on a sized gel;

[0069] d) contacting the resulting gel or the obtained nucleic acidmolecule with a nucleic acid probe labelled with a detectable marker andcapable of specifically hybridizing to said nucleic acid molecule (saidhybridization being made in stringent hybridization conditions);

[0070] e) detecting labelled bands or the in situ nucleic acid moleculeswhich have hybridized to the said nucleic acid molecule labelled with adetectable marker to create a unique band pattern or an in situ markingspecific to the subject;

[0071] f) preparing other nucleic acid molecules encoding the peptideaccording to the invention obtained from the cells of other patients fordiagnosis by step a-e; and

[0072] g) comparing the unique band pattern specific to the nucleic acidmolecule of subjects suffering from the disorder from step e and thenucleic acid molecule obtained for diagnosis from step f to determinewhether the patterns are the same or different and to diagnose thereby apredisposition or a resistance to the disorder if the patterns are thesame or different.

[0073] The present invention is also related to a method for diagnosinga predisposition or a resistance to a disorder associated with theactivity of a specific allele of the peptide according to the inventionor the presence of said peptide at the surface of cells and/orassociated with infectious agents such as HIV viruses present in asubject. Said method comprises:

[0074] a) obtaining a sample of a body fluid, preferably a blood samplecomprising antigen presenting cells, from a subject;

[0075] b) adding to said sample a ligand and/or an anti-ligand accordingto the invention;

[0076] c) detecting the cross-reaction between said ligand and/or saidanti-ligand and the specific peptide according to the invention; and

[0077] d) determining whether the peptide corresponds to a receptor oran inactive receptor according to the invention and diagnosing thereby apredisposition or a resistance to the disorder according to the type ofthe peptide present in the body fluid of the subject.

[0078] The present invention concerns also a diagnostic and/or dosagedevice, preferably a kit, comprising the peptides, the nucleic acidmolecules, the nucleic acid probes, the ligands and/or the anti-ligandsaccording to the invention, their portions (such as primers, probes,epitopes, . . . ) or a mixture thereof, being possibly labelled with adetectable marker.

[0079] Said diagnostic and/or dosage device comprises also the reactantsfor the detection and/or the dotage of antigens, antibodies or nucleicacid sequences through a method selected from the group consisting of insitu hybridization, hybridization or recognition by marked specificantibodies, specially ELISA® (Enzyme Linked Immunosorbent Assay) or RIA®(Radio Immunoassay), methods on filter, on a solid support, in solution,in “sandwich”, on gel, by Dot blot hybridization, by Northern blothybridization, by Southern blot hybridization, by isotopic ornon-isotopic labelling (such as immunofluorescence or biotinylation), bya technique of cold probes, by genetic amplification, particularly PCR,LCR, NASBA or CPR, by a double immunodiffusion, by acounter-immunoelectrophoresis, by haemagglutination and/or a mixturethereof.

[0080] A last aspect of the present invention concerns a method ofpreparing peptides according to the invention, which comprises

[0081] a) constructing a vector adapted for expression in a cell whichcomprises the regulatory elements necessary for the expression ofnucleic acid molecules in the cell operatively linked to nucleic acidmolecule encoding said peptide so as to permit expression thereof,wherein the cell is preferably selected from the group consisting ofbacterial cells, yeast cells, insect cells and mammalian cells;

[0082] b) inserting the vector of step a in a suitable host cell;

[0083] c) incubating the cell of step b under conditions allowing theexpression of the peptide according to the invention;

[0084] d) recovering the peptide so obtained; and

[0085] e) purifying the peptide so recovered, thereby preparing anisolated peptide-according to the invention.

[0086] The deposits of micro-organisms AchCCR5-SAB1A7 and CHO-K1-PEFINHCCR5-1/16 were made according to the Budapest Treaty in the BelgiumCoordinated Collection of Micro-organisms (BCCM), Laboratorium voorMoleculaire Biologic (LMBP), Universiteit Gent, K. L. Ledeganckstraat35, B-9000 GENT, BELGIUM.

BRIEF DESCRIPTION OF THE FIGURES

[0087]FIG. 1 represents the primary structure of the peptides accordingto the invention.

[0088]FIG. 2 represents the amino acids sequence of the active humanCCR5 chemokine receptor according to the invention aligned with that ofthe human CCR1, CCR2b, CCR3 and CCR4 receptors. Amino acids identicalwith the active CCR5 sequence are boxed.

[0089]FIG. 3 shows the chromosomal organisation of the human CCR2 andCCR5 chemokine receptor genes.

[0090]FIG. 4 shows the functional expression of the human active CCR5receptor in a CHO-K1 cell line.

[0091]FIG. 5 represents the distribution of MRNA encoding the CCR5receptor in a panel of human cell lines of haematopoietic origin.

[0092]FIG. 6 represents the structure of the mutant form of human CCR5receptor.

[0093]FIG. 7 represents the quantification of ENV proteins-mediatedfusion by luciferase assays.

[0094]FIG. 8 represents genotyping of individuals by PCR and segregationof the CCR5 alleles in CEPH families.

[0095]FIG. 9 represents the FACS analysis of sera anti-CCR5 on aCCR5-CHO cell line according to the invention.

[0096]FIG. 10 represents the inhibition of HIV infectivity withanti-CCR5 antibodies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. Experimentals

[0097] Materials

[0098] Recombinant human chemokines, including MCP-1, MIP-1α, MIP-1β,RANTES, IL-8 and GROα were obtained from R & D Systems (London, UK).[¹²⁵I]MIP-1α (specific activity, 2200 Ci/mmol) was obtained from DupontNEN (Brussels, Belgium). Chemokines obtained from R & D Systems werereported by the supplier as >97% pure on SDS-PAGE (sodium dodecylsulphate-polyacrylamide gel electrophoresis) and biologically active ona bioassay specific for each ligand. The lyophilised chemokines weredissolved as a 100 μg/ml solution in a sterile phosphate-buffered saline(PBS) and this stock solution was stored at −20° C. in aliquots.Chemokines were diluted to the working concentration immediately beforeuse. All cell lines used in the present study were obtained from theATCC (Rockville, MID, USA).

[0099] Cloning and Sequencing

[0100] The mouse MOP020 clone was obtained by low stringency polymerasechain reaction, as described previously [24, 34], using genomic DNA astemplate. A human genomic DNA library (Stratagene, La Jolla, Calif.),constructed in the lambda DASH vector was screened at low stringency[39] with the MOP020 (511 bp) probe. The positive clones were purifiedto homogeneity and analysed by Southern blotting. The restriction map ofthe locus was determined and a relevant XbaI fragment of 4,400 bp wassubcloned in pBluescript SK+ (Stratagene). Sequencing was performed onboth strands after subcloning in M13mp derivatives, using fluorescentprimers and an automated DNA sequencer (Applied Biosystem 370A).Sequence handling and data analysis was carried out using theDNASIS/PROSIS software (Hitachi), and the GCG software package (GeneticsComputer Group, Wisconsin).

[0101] Expression in Cell Lines

[0102] The entire coding region was amplified by PCR as a 1056 bpfragment, using primers including respectively the BamHI and XbaIrecognition sequences, and cloned after restriction in the correspondingsites of the eukaryotic expression vector pcdna3 (Invitrogen, San Diego,Calif.). The resulting construct was verified by sequencing, andtransfected in CHO-K1 cells as described [35]. Two days aftertransfection, selection for stably transfected cell lines was initiatedby the addition of 400 μg/ml G418 (Gibco), arid resistant clones wereisolated at day 10. CHO-K1 cells were cultured using Ham's F12 medium,as previously described [35, 11]. The expression of the active CCR5receptor in the various cell clones was evaluated by measuring thespecific transcript level by Northern blotting, on total RNA preparedfrom the cells (see below).

[0103] Binding Assays

[0104] Stably transfected CHO-K1 cells expressing the active CCR5receptor were grown to confluence and detached from culture dishes byincubation in phosphate-buffered saline (PBS) supplemented with 1 MmEDTA. Cells were collected by low speed centrifugation and counted in aNeubaeur cell. Binding assays were performed in polyethylene minisorptubes (Nunc) in a final volume of 200 μl PBS containing 0.2% bovineserum albumin (BSA) and 10⁶ cells, in presence of [¹²⁵I]-MIP-1α. Nonspecific binding was determined by addition of 10 Nm unlabelled MIP-1α.The concentration of labelled ligand was 0.4 Nm (around 100 000 cpm pertube). The incubation was carried out for 2 hours at 4° C., and was:stopped by the rapid addition of 4 ml ice-cold buffer, and immediatecollection of cells by vacuum filtration through GF/B glass fiberfilters (Whatmann) pre-soaked in 0.5% polyethyleneinimine (Sigma).Filters were washed three times with 4 ml ice-cold buffer and counted ina gamma counter.

[0105] Biological Activity

[0106] The CHO-K1 cell lines stably transfected with the pcdna3/CCR5construct or wild type CHO-K1 cells (used as controls) were plated ontothe membrane of Transwell cell capsules (Molecular Devices), at adensity of 2.5 10⁵ cells/well in Ham's F12 medium. The next day, thecapsules were transferred in a microphysiometer (Cytosensor, MolecularDevices), and the cells were allowed to equilibrate for approximatelytwo hours by perfusion of 1 Mm phosphate-buffered (Ph 7.4) RPMI-1640medium containing 0.2% BSA. Cells were then exposed to variouschemokines diluted in the same medium, for a 2 mm duration.Acidification rates were measured at one minute intervals.

[0107] Northern Blotting

[0108] Total RNA was isolated from transfected CHO-K1 cell lines, from apanel of human cell lines of haematopoietic origin and from a panel ofdog tissues, using the RNeasy kit (Qiagen). RNA samples (10 μg per lane)were denatured in presence of glyoxal [26], fractionated on a 1% agarosegel in a 10 Mm phosphate buffer (Ph 7.0), and transferred to nylonmembranes (Pall Biodyne A, Glen Cove, N.Y.) as described [42]. Afterbaking, the blots were prehybridized for 4 h at 42° C. in a solutionconsisting of 50% formamide, 5× Denhardt solution (1× Denhardt: 0.02%Ficoll, 0.02% polyvinylpyrolidone, 0.02% BSA), 5×SSPE (1×SSPE: 0.18 MNaCl, 10 Mm Na phosphate, 1 Mm EDTA Ph 8.3), 0.3% Sodium DodecylSulphate (SDS), 250 μg per ml denatured DNA from herring testes. DNAprobes were (α³²P)-labelled by random priming [14]. Hybridizations werecarried out for 12 h at 42° C. in the same solution containing 10%(wt/vol) dextran sulphate and the heat denatured probe. Filters werewashed up to 0.1×SSMC (1×SSC: 150 Mm NaCl, 15 Mm Na Citrate Ph 7.0),0.1% SDS at 60° C. and autoradiographed at −70° C. using Amersham β-maxfilms.

2. Results and Discussion

[0109] Cloning and Structural Analysis

[0110] The sequence homology characterising genes encoding Gprotein-coupled receptors has allowed the cloning by low stringencypolymerase chain reaction (PCR) of new members of this gene family [24,34]. One of the clones amplified from mouse genomic DNA, named MOP020presented strong similarities with characterised chemokine receptors,sharing 80% identity with the MCP-1 receptor (CCR2) [8], 65% identitywith the MIP-1α/RANTES receptor (CCR1) [31], and 51% identity with IL-8receptors [20, 30]. The clone was used as a probe to screen a humangenomic library. A total of 16 lambda phage clones were isolated. It wasinferred from the restriction pattern of each clone and from partialsequence data that all clones were belonging to a single contig in whichtwo different coding sequences were included. One of the codingsequences was identical to the reported CDNA encoding the CCR2 receptor[8, 44]. A 4.400 pb XbaI fragment of a representative clone containingthe second region of hybridization was subcloned in Pbluescript SK+.Sequencing revealed a novel gene, tentatively named CCR5, sharing 84%identity with the MOP020 probe, suggesting that MOP020 is the mouseortholog of CCR5. MOP020 does not correspond to any of the three mousechemokine receptor genes cloned recently [16], demonstrating theexistence of a fourth murine chemokine receptor.

[0111] The sequence of CCR5 revealed a single open reading frame of 352codons encoding a protein of 40,600 Da. The sequence surrounding theproposed initiation codon is in agreement with the consensus asdescribed by Kozak [22], since the nucleotide in −3 is a purine. Thehydropathy profile of the deduced amino acid sequence is consistent withthe existence of 7 transmembrane segments. Alignment of the CCR5 aminoacid sequence with that of other functionally characterised humanCC-chemokine receptors is represented in FIG. 2. The highest similarityis found with the CCR2 receptor [8] that shares 75.8% identicalresidues. There is also 56.3% identity with the CCR1 receptor [31],58.4% with the CCR3 [10], and 49.1% with the CCR4 [37]. CCR5 representstherefore a new member of the CC-chemokine receptor group [30]. Like therelated CCR1 and IL-8 receptors [20, 29, 31, 16] the coding region ofCCR5 appears as intronless. From our partial sequencing data, the CCR2gene is also devoid of intron in the first two thirds of its codingsequence.

[0112] Sequence similarities within the chemokine receptor family arehigher in the transmembrane-spanning domains, and in intracellularloops. As an example, the identity score between CCR5 and CCR2 goes upto 92% when considering the transmembrane segments only. Lowersimilarities are found in the N-terminal extracellular domain, and inthe extracellular loops. The N-terminal domain of the IL-8 and CCR2receptors has been shown to be essential for interaction with the ligand[19, 18]. The variability of this region among CC-chemokine receptorspresumably contributes to the specificity towards the various ligands ofthe family.

[0113] A single potential site for N-linked glycosylation was identifiedin the third extracellular loop of CCR5 (FIG. 1). No glycosylation sitewas found in the N-terminal domain of the receptor, where most Gprotein-coupled receptors are glycosylated. The other chemokinereceptors CCR1 and CCR2 present such an N-linked glycosylation site intheir N-terminal domain [31, 8]. By contrast, the CCR3 receptor [10]does not display glycosylation sites neither in the N-terminus, nor inextracellular loops. The active CCR5 receptor has four cysteines in itsextracellular segments, and all four are conserved in the other CC— andCXC-chemokine receptors (FIG. 2). The cysteines located in the first andsecond extracellular loops are present in most G protein-coupledreceptors, and are believed to form a disulphide bridge stabilising thereceptor structure [41]. The two other cysteines, in the N-terminalsegment, and in the third extracellular loop could similarly form astabilising bridge specific to the chemokine receptor family. Theintracellular domains of CCR5 do not include potential sites forphosphorylation by protein kinase C (PKC) or protein kinase A. PKCsites, involved in heterologous desensitisation are frequent in thethird intracellular loop and C-terminus of G protein-coupled receptors.CCR1 is also devoid of PKC sites. In contrast, all CC-chemokinereceptors, are rich in serine and threonine residues in the C-terminaldomain. These residues represent potential phosphorylation sites by thefamily of G protein-coupled receptor kinases, and are probably involvedin homologous desensitisation [41]. Five of these S/T residues areperfectly aligned in all five receptors (FIG. 2).

[0114] Physical Linkage of the CCR5 and CCR2 Genes

[0115] As stated above, the 16 clones isolated with the MOP020 probecorresponded to a single contig containing the CCR5 and CCR2 genes. Theorganisation of this contig was investigated in order to characterisethe physical linkage of the two receptor genes in the human genome. Acombination of restriction mapping, Southern blotting, fragmentsubcloning and partial sequencing allowed to determine the respectiveborders and overlaps of all clones. Out of the 16 clones, 9 turned outto be characterised by a specific restriction map, and theirorganisation is depicted in FIG. 3. Four of these clones (#11, 18, 21,22) contained the CCR2 gene alone, four clones (# 7, 13, 15, 16)contained the ChemR13 gene alone and one clone (#9) contains part ofboth coding sequences. The CCR2 and CCR5 genes are organised in tandem,CCR5 being located downstream of CCR2. The distance separating CCR2 andCCR5 open reading frames is 17.5 kb. The chromosomal localisation of thetandem is presently unknown. Other chemokine receptors have however beenlocated in the human genome: the CCR1 gene was localised by fluorescencein situ hybridization to the p21 region of human chromosome 3 [16]. Thetwo IL-8 receptor genes, and theft pseudogene have been shown to beclustered on the human 2q34-q35 region [1].

[0116] Functional Expression and Pharmacology of the Active CCR5Receptor

[0117] Stable CHO-K1 cell lines expressing the active CCR5 receptor wereestablished and were screened on the basis of the level of CCR5transcripts as determined by Northern blotting. Three clones wereselected and tested for biological responses in a microphysiometer,using various CC— and CXC-chemokines as potential agonists. Wild typeCHO-K1 dells were used as control to ensure that the observed responseswere specific for the transfected receptor, and did not result from theactivation of endogenous receptors. The microphysiometer allows the realtime detection of receptor activation, by measuring the modifications ofcell metabolism resulting from the stimulation of intracellular cascades[33]. Several studies have already demonstrated the potential ofmicrophysiometry in the field of chemokine receptors. Modifications ofmetabolic activity in human monocytes, in response CC-chemokines, weremonitored using this system [43]. Similarly, changes in theacidification rate of THP-1 cells (a human monocytic cell line) inresponse to MCP-1 and MCP-3 have been measured [36]. The estimation ofthe EC₅₀ for both proteins, using this procedure, was in agreement withthe values obtained by monitoring the intracellular calcium in otherstudies [8, 15].

[0118] Ligands belonging to the CC— and CXC-chemokine classes weretested on the CCR5 transfected CHO-K1 cells. Whereas MIP-1α, MIP-1β andRANTES were found to be potent activators of the new receptor (FIG. 4),the CC-chemokines MCP-1, MCP-2 and MCP-3, and the CXC-chemokines GROαand IL-8 had no effect on the metabolic activity, even at the highestconcentrations tested (30 Nm). The biological activity of one of thechemokines inducing no response on CCR5 (IL-8) could be demonstrated ona CHO-K1 cell line transfected with the IL-8A interleukin receptor(Mollereau et al., 1993): IL-8 produced a 160% increase in metabolicactivity as determined using the microphysiometer. The biologicalactivity of the MCP-2 and MCP-3 preparations as provided by J. Van Dammehave been widely documented [2, 40]. MIP-1α, MIP-1β and RANTES weretested on the wild type CHO-K1 cells, at a 30 Nm-concentration, and noneof them induced a metabolic response. On the CCR5 transfected CHO-K1cell line, all three active ligands (MIP-1α, MIP-1β and RANTES) caused arapid increase in acidification rate, reaching a maximum by the secondor third minute after perfusion of the ligand. The acidification ratereturned to basal level within 10 minutes. The timing of the cellularresponse is similar to that observed for chemokines on their naturalreceptors in human monocytes [43]. When agonists were applied repeatedlyto the same cells, the response was strongly reduced as compared to thefirst stimulation, suggesting the desensitisation of the receptor. Allmeasurements were therefore obtained on the first stimulation of eachcapsule.

[0119] The concentration-effect relation was evaluated for the threeactive ligands in the 0.3 to 30 Nm range (FIGS. 3B and C). The rankorder of potency was MIP-1α>MIP-1β=RANTES. At 30 Nm concentrations, theeffect of MIP-1α appeared to saturate (at 156% of baseline level) whileMIP-1β and RANTES were still in the ascending phase. Higherconcentrations of chemokines could however not be used. The EC50 wasestimated around 3 Nm for MIP-1α. The concentrations necessary forobtaining a biological response as determined by using themicrophysiometer are in the same range as those measured byintracellular calcium mobilisation for the CCR1 [31], the CCR2A and B[8], and the CCR3 [10] receptors. The ligand specificity of CCR5 issimilar to that reported for CCR3 [10]. CCR3 was described as the firstcloned receptor responding to MIP-1β. However, MIP-1β at 10 Nm elicits asignificant effect on the CCR5, while the same concentration is withouteffect on the CCR3 transfected cells [10]. These data suggest that CCR5could be a physiological receptor for MIP-1β.

[0120] Binding experiments using [¹²⁵I]-human MIP-1α as ligand did notallow to demonstrate specific binding to CCR53 expressing CHO-K1 cells,using as much as 0.4 Nm radioligand and 1 million transfected cells pertube. Failure to obtain binding data could be attributed to a relativelylow affinity of the receptor for MIP-1α.

[0121] Northern Blotting Analysis

[0122] Northern blotting performed on a, panel of dog tissues did notallow to detect transcripts for CCR5. Given the role of the chemokinereceptor family in mediating chemoattraction and activation of variousclasses of cells involved in inflammatory and immune responses, theprobe was also used to detect specific transcripts in a panel of humancell lines of haematopoietic origin (FIG. 5). The panel includedlymphoblastic (Raji) and T lymphoblastic (Jurkat) cell lines,promyeloblastic (KG-1A) and promyelocytic (HL-60) cell lines, amonocytic (THP-1) cell line, an erythroleukemia (HEL 92.1.7) cell line,a megakaryoblastic (MEG-01) cell line, and a myelogenous leukaemia(K-562) cell line. Human peripheral blood mononuclear cells (PBMC),including mature monocytes and lymphocytes, were also tested. CCR5transcripts (4.4 kb) could be detected only in the KG-1A promyeloblasticcell line, but were not found in the promyelocytic cell line HL-60, inPBMC, or in any of the other cell lines tested. These results suggestthat the active CCR5 receptor could be expressed in precursors of thegranulocytic lineage. CC-chemokines have been reported to stimulatemature granulocytes [27, 38, 23, 2]. However, recent data have alsodemonstrated a role of CC— and CXC-chemokines in the regulation of mouseand human myeloid progenitor cell proliferation [6, 7].

[0123] CCR5 was shown to respond to MIP-1α, MIP-1β and RANTES, the threechemokines identified as the major HIV-suppressive factors produced byCD8⁺ T cells [9], and released in higher amounts by CD4⁺ T lymphocytesfrom uninfected but multiply exposed individuals [51]. CCR5 represents amajor co-receptor for macrophage-tropic (M-tropic) HIV-1 primaryisolates and strains [45, 50]. M-tropic strains predominate during theasymptomatic phase of the disease in infected individuals, and areconsidered as responsible for HIV-1 transmission. Strains adapted forgrowth in transformed T-cell lines (T-tropic strains) use as aco-receptor LESTR (or fusin) [50], an orphan receptor also belonging tothe chemokine receptor family, but not yet characterized functionally[21, 52, 53]. Dual-tropic viruses, which may represent transitionalforms of the virus in late stages of infection [54] are shown to useboth CCR5 and LESTR as co-receptors, as well as the CC-chemokinereceptors CCR2b and CCR3 [47]. The broad spectrum of co-receptor usageof dual-tropic viruses suggests that within infected individuals, thevirus may evolve at least in part from selection by a variety ofco-receptors expressed on different cell types.

[0124] Identification of an Inactive ΔCCR5 Receptor

[0125] It is known that some individuals remain uninfected despiterepeated exposure to HIV-1 [55, 56, 51]. A proportion of theseexposed-uninfected individuals results from the relatively low risk ofcontamination after a single contact with the virus, but it has beenpostulated that truly resistant individuals do exist. In fact, CD4⁺lymphocytes isolated from exposed-uninfected individuals are highlyresistant to infection by primary M-tropic, but not T-tropic HIV-1strains. Also, peripheral blood mononuclear cells (PBMC) from differentdonors are not infected equally with various HIV-1 strains [57-59].Given the key role played by CCR5 in the fusion event that mediatesinfection by M-tropic viruses, it is postulated that variants of CCR5could be responsible for the relative or absolute resistance to HIV-1infection exhibited by some individuals, and possibly for thevariability of disease progression in infected patients [66]. TheInventors selected three HIV-1 infected patients known to be slowprogressors, and four seronegative individuals as controls; the fullcoding region of their CCR5 gene was amplified by PCR and sequenced.Unexpectedly, one of the slow progressors, but also two of theuninfected controls, exhibited heterozygosity at the CCR5 locus for abiallelic polymorphism. The frequent allele corresponded to thepublished CCR5 sequence, while the minor one displayed a 32 bp deletionwithin the coding sequence, in a region corresponding to the secondextracellular loop of the receptor (FIG. 6). The FIG. 6 is the structureof the mutant form of human CC-chemokine receptor 5. α, The amino acidsequence of the nonfunctional Δccr5 protein is represented. Thetransmembrane organization is given by analogy with the predictedtransmembrane structure of the wild-type CCR5. Amino acids representedin black correspond to unnatural residues resulting from the frame shiftcaused by the deletion. The mutant protein lacks the last threetransmembrane segments of CCR5, as well as the regions involved in Gprotein-coupling. β, Nucleotide sequence of the CCR5 gene surroundingthe deleted region, and translation into the normal receptor (top) orthe truncated mutant (ccr5, bottom). The 10-bp direct repeat isrepresented in italics. The full size .coding region of the CCR5 genewas amplified by PCR, using 5′-TCGAGGATCCAAGATGGATTATCAAGT-3′ and5′-CTGATCTAGAGCCATGTGCACAACTCT-3′ as forward and reverse primersrespectively. The PCR products were sequenced on both strands using thesame oligonucleotides as primers, as well as internal primers, andfluorochrome-labeled dideoxynucleotides as terminators. The sequencingproducts were run on an Applied Biosystem sequencer, and ambiguouspositions were searched along the coding sequence. When the presence ofa deletion was suspected from direct sequencing, the PCR products werecloned after restriction with BamHI and XbaI endonucleases into pcdna3.Several clones were sequenced to confirm the deletion. The deletion wasidentical in three unrelated individuals investigated by sequencing.

[0126] Cloning of the PCR product and sequencing of several clonesconfirmed the deletion. The deletion causes a frame shift, which isexpected to result in premature termination of translation. The proteinencoded by this mutant allele (Δccr5) therefore lacks the last threetransmembrane segments of the receptor. A 10-bp direct repeat flankingthe deleted region (FIG. 6b) on both sides is expected to have promotedthe recombination event leading to the deletion. Numerous mutagenesisstudies performed on various classes of G protein-coupled receptors,including chemokine receptors, makes it clear that such a truncatedprotein is certainly not functional in terms of chemokine-induced signaltransduction: it lacks the third intracellular loop and C-terminalcytoplasmic domains, the two regions involved primarily in G proteincoupling [41]. In order to test whether the truncated protein was ableto function as a HIV-1 co-receptor, the Inventors tested its ability tosupport membrane fusion by both primary M-tropic and dual-tropic virusENV proteins. The recombinant protein was expressed in quail QT6 cellstogether with human CD4. The QT6 cells were then mixed with HeLa cellsexpressing the indicated viral ENV protein and the extent of cell-cellfusion measured using a sensitive and quantitative gene-reporter assay.In contrast to wild-type CCR5, the truncated receptor did not allowfusion with cells expressing the ENV protein from either M-tropic ordual-tropic viruses (FIG. 7). The FIG. 7 represents the quantificationof ENV protein-mediated fusion by luciferase assay. To quantifycell-cell fusion events, Japanese quail QT6 fibrosarcoma cells weretransfected or cotransfected as indicated with the pcdna3 vector(Invitrogen) containing the coding sequence for wild-type CCR5, thetruncated ccr5 mutant, the CCR2b or the Duffy chemokine receptors, orwith the PCDNA3 vector alone. The target cells were also transfectedwith human CD4 expressed from the CMV promoter and the luciferase geneunder the control of the T7 promoter. HeLa effector cells were infected(MOI=10) with vaccinia vectors expressing T7-polymerase (vTF1.1) andeither the JR-FL (vCB28) or 89.6 (vBD3) envelope proteins. Theluciferase activity resulting from cell fusion is expressed as thepercentage of the activity (in relative light units) obtained forwild-type CCR5. All transfections were performed with an identicalquantity of plasmid DNA using pcdna3 as carrier when necessary. Toinitiate fusion, target and effector cells were mixed in 24 well platesat 37° C. in the presence of ara-C and rifampicin, and allowed to fusefor 8 hours. Cells were lysed in 150 μl of reporter lysis buffer(Promega) and assayed for luciferase activity according to themanufacturer's instructions (Promega).

[0127] Coexpression of Δccr5 with wild-type CCR5 consistently reducedthe efficiency of fusion for both JR-FL and 89.6 envelopes, as comparedwith CCR5 alone. Whether this in vitro inhibitory effect (not shared bythe chemokine receptor Duffy, used as control) also occurs in vivo ispresently not known. Coexpression with the CCR2b receptor [31], which isthe CC-chemokine receptor most closely related to CCR5 but does notpromote fusion by M-tropic HIV-1 strains [48], did not rescue themutation by formation of a hybrid molecule (FIG. 7).

[0128] The FIG. 8 represents genotyping of individuals by PCR andsegregation of the CCR5 alleles in CEPH families, α, Autoradiographyillustrating the pattern resulting from PCR amplification and EcoRIcleavage for individuals homozygous for the wild-type CCR5 allele(CCR5/CCR5), the null Δccr5 allele (Δccr5/Δccr5), and for heterozygotes(CCR5/Δccr5). A 735 bp PCR product is cleaved into a common band of 332bp for both alleles, and into 403 and 371 bp bands for the wild-type andmutant alleles, respectively. b, Segregation of the CCR5 alleles in twoinformative families of the CEPH. Half-black and white symbols representheterozygotes and wild-type homozygotes, respectively. For a fewindividuals in the pedigrees, DNA was not available (ND: notdetermined). PCRs were performed on genomic DNA samples, using5′-CCTGGCTGTCGTCCATGCTG-3′ and 5′-CTGATCTAGAGCCATGTGCACAACTCT-3′ asforward and reverse primers respectively. Reaction mixtures consisted in30 μl of 10 Mm Tris-Hcl buffer Ph 8.0, containing 50 Mm Kcl, 0.75 MmMgCl₂, 0.2 Mm dCTP, dGTP and dTTP, 0.1 Mm dATP, 0.5 μi [α-³²P]-DATP,0.01% gelatine, 5% DMSO, 200 ng target DNA, 60 ng of each of the primersand 1.5 U Taq polymerase. PCR conditions were: 93° C. for 2 min 30; 93°C. for 1 min, 60° C. for 1 min, 72° C. for 1 min, 30 cycles; 72° C. for6 min. After the PCR reaction, the samples were incubated for 60 mm at37° C. with 10 U EcoRI, and 2 μl of the denatured reaction mixture wasapplied onto a denaturing 5% polyacrylamide gel containing 35% formamideand 5.6 M urea. Bands were detected by autoradiography.

[0129] Based on the 14 chromosomes tested in the first experiment, thedeleted Δccr5 allele appeared rather frequent in the Caucasianpopulation. The accurate frequency was further estimated by testing(FIG. 8a) a large cohort of Caucasian individuals, including unrelatedmembers of the CEPH (Centre d'Etude des Polymorphismes Humains)families, part of the IRIBHN staff, and a bank of anonymous DNA samplesfrom healthy individuals collected by the Genetics Department of theErasme Hospital in Brussels. From a total of more than 700 healthyindividuals, the allele frequencies were found to be 0.908 for thewild-type allele, and 0.092 for the mutant allele (Table I). Thegenotype frequencies observed in the population were not significantlydifferent from the expected Hardy-Weinberg distribution (CCR5/CCR5:0.827 vs 0.824; CCR5/Δccr5: 0.162 vs 0.167; Δccr5/Δccr5: 0.011 vs 0.008,p>0.999), suggesting that the null allele has no drastic effect onfitness. Using two informative CEPH families, it was confirmed that—thewild-type CCR5 gene and its Δccr5 variant were allelic, and segregatedin a normal mendelian fashion (FIG. 8b). Interestingly, a cohort of 124DNA samples originating from Central Africa (collected from Zaire,Burkina Fasso, Cameroun, Senegal and Benin) and Japan did not reveal asingle Δccr5 mutant allele, suggesting that this allele is either absentor very rare in Asian, African black populations (Table I).

[0130] The consequences of the existence of a null allele of CCR5 in thenormal Caucasian population were then considered in terms ofsusceptibility to infection by HIV-1. If, as it is predicted, CCR5 playsa major (not redundant) role in the entry of most primary virus strainsinto cells, then Δccr5/Δccr5 individuals should be particularlyresistant to HIV-1 challenge, both in vitro and in vivo. The frequencyof the Δccr5/Δccr5 genotype should therefore be significantly lower inHIV-1 infected patients, and increased in exposed-uninfectedindividuals. Also, if heterozygotes have a statistical advantage due tothe lower number of functional receptors on their white blood cells, orto the possible dominant-negative properties of the mutant allele, thefrequency of heterozygotes (and mutant alleles) should be decreased inHIV-infected populations. These hypotheses were tested by genotyping alarge number of seropositive Caucasian individuals (n=645) belonging tocohorts originating from various hospitals from Brussels, Liège andParis (Table I). Indeed, it was found that within this large series, thefrequency of the null Δccr5 allele was significantly reduced from 0.092to 0.053 (p<10⁻⁵). The frequency of heterozygotes was also reduced from0.162 to 0.106 (p<0.001) and not a single Δccr5/Δccr5 individual couldbe found (p<0.01).

[0131] Altogether, functional and statistical data suggest that CCR5 isindeed the major co-receptor responsible for natural infection byM-tropic HIV-1 strains. Individuals homozygous for the null Δccr5 allele(about 1% of the Caucasian population) have apparently a strongresistance to infection. It is unclear at this point whether resistanceto HIV-1 is absolute or relative, and whether resistance will varydepending on the mode of viral contamination. Larger cohorts ofseropositive individuals will have to be tested in order to clarify thispoint. Heterozygotes have a milder though significant advantage:assuming an equal probability of contact with HIV, it can be inferredfrom Table I that heterozygotes have a 39% reduction in their likelinessof becoming seropositive, as compared to individuals homozygous for thewild-type CCR5 allele. Both a decrease in functional CCR5 receptornumber, and a dominant-negative effect of Δccr5 in vivo, comparable towhat is observed in the in vitro experiments (FIG. 7) are possibleexplanations for this relative protection. The mutant allele, which canbe regarded as a natural knock-out in human, is not accompanied by anobvious phenotype in homozygous individuals. Nevertheless, the lack ofovert phenotype, taken together with the relative protection thatcharacterizes heterozygous subjects, suggests that pharmacologicalagents that selectively block the ability of HIV-1 to utilize CCR5 as acofactor, could be effective in preventing HIV-1 infection, and would bepredicted not be associated with major side effects resulting from CCR5inactivation. These pharmaceutical agents could be used with othercompounds which are able to block other chemokine .receptors used asco-receptors by some HIV-primary isolates in order to infect other cells[47]. The prevalence of the null allele in the Caucasian populationraises the question of whether pandemia of HIV (or related viruses usingthe same co-receptor) have contributed during mankind's evolution tostabilize by selection the mutant ccr5 allele at such a high frequency.

[0132] Production of Antibodies Anti-CCR5

[0133] Antibodies were produced by genetic immunisation. Six week oldfemales balb/c mice were used. DNA coding for the human CCR5 receptorwas inserted in the expression vector pcdna3 under the control of theCMV promotor and 100 μg DNA was injected in the anterior tibial muscle,five days after pre-treatment of this muscle with cardiotoxine (fromvenom of Naja Nigricolis). Injections were repeated twice at three weekintervals. Fifteen days after the last injection, blood was taken fromeach animal and sera were tested for the presence of anti-CCR5antibodies.

[0134] Test of Sera Using Fluorescence Activated Cell Sorter (FACS)

[0135] Sera were tested by fluorescence activated cell sorting usingrecombinant CHO cells expressing the CCR5 receptor. Briefly, cells weredetached using a PBS-EDTA-EGTA solution and incubated into PBS-BSAmedium for 30 minutes at room temperature with 5 μl serum on the basisof 100,000 cells per tube. Cells were then washed and incubated for 30minutes in ice together with anti-mouse antibody labelled withfluorescein. Cells were washed, taken up into 200 μl of a PBS-BSAsolution and fluorescence was analysed by FACS (FACSCAN,Becton-Dickinson). 10,000 cells were counted. Wild type CHO orrecombinant CHO cells expressing the human CCR2b receptor were used ascontrols.

[0136] When tested by FACS analysis 2 weeks after the last injection(FIG. 9), all the sera from mice immunised with CCR5 CDNA, clearlyrecognised the native receptor expressed on CHO cells (mean offluorescence=200), without significant cross reaction with control cellsexpressing CCR2b (mean of fluorescence=20).

[0137] Sera were tested on either a CHO cell line expressing high levelof CCR5 receptor (black histogram) or a CHO cell line expressing CCR2breceptor (white histogram) as negative control. Each serum was testedindividually.

[0138] Antibodies Anti-CCR5 and HIV Infectivity

[0139] Peripheral blood mononuclear cells (PBMC) from one donorhomozygous from wild type CCR5 gene, were isolated and cultivated 3 daysin presence of PHA.

[0140] On day 4, 800 μl of cells (10⁵ cells/ml) were incubated with 8 μlof sera from mice immunised with CCR5 CDNA, 30 minutes at 37° C. 1 ml ofviral solution (JRCSF HIV strain) is then added and incubated during 2hours. Cells were then washed twice and cultivated during 15 days.

[0141] Aliquot of medium is taken at days 0, 4, 7, 10 and 14 and thedosage of antigen p24 is performed.

[0142] 14 days after the beginning of the experiment, one serum (serumB0) totally block the production of p24, indicating its ability to blockthe infection of the lymphocytes by this HIV strain (FIG. 10). Otherserums also exhibit a partial or total effect on this infection (serumA2 and B1). All the other sera did not show any effect on thisinfection.

[0143] Production of Monoclonal Antibodies

[0144] Mice with the highest title of CCR5 antibodies were selected formonoclonal antibodies production and injected intravenously with 10⁷recombinant CHO-K1 cells expressing human CCR5 receptors. Three dayslater, animals were sacrificed and fusion of splenic cells or cells fromlymph nodes near the site of injection with SP2/0 myeloma cells, wereperformed. Fusion protocol used was that of Galfre et al. (Nature 266,550 (1977)). A selective HAT (hypoxanthine/aminopterin/thymidin) mediumis used to select hybridomas and their supernatants are tested by FACSusing recombinant CHO cells expressing the human CCR5 receptor, as itwas done for the sera. Positives hybridomas are then cloned by limiteddilution. Clones that are shown positive by FACS analyses are thenexpanded and produced in ascites in balb/C mice.

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1 18 1 792 DNA Homo sapiens 1 gaattccccc aacagagcca agctctccatctagtggaca gggaagctag cagcaaacct 60 tcccttcact acaaaacttc attgcttggccaaaaagaga gttaattcaa tgtagacatc 120 tatgtaggca attaaaaacc tattgatgtataaaacagtt tgcattcatg gagggcaact 180 aaatacattc taggacttta taaaagatcactttttattt atgcacaggg tggaacaaga 240 tggattatca agtgtcaagt ccaatctatgacatcaatta ttatacatcg gagccctgcc 300 aaaaaatcaa tgtgaagcaa atcgcagcccgcctcctgcc tccgctctac tcactggtgt 360 tcatctttgg ttttgtgggc aacatgctggtcatcctcat cctgataaac tgcaaaaggc 420 tgaagagcat gactgacatc tacctgctcaacctggccat ctctgacctg tttttccttc 480 ttactgtccc cttctgggct cactatgctgccgcccagtg ggactttgga aatacaatgt 540 gtcaactctt gacagggctc tattttataggcttcttctc tggaatcttc ttcatcatcc 600 tcctgacaat cgataggtac ctggctgtcgtccatgctgt gtttgcttta aaagccagga 660 cggtcacctt tggggtggtg acaagtgtgatcacttgggt ggtggctgtg tttgcgtctc 720 tcccaggaat catctttacc agatctcaaaaagaaggtct tcattacacc tgcagctctc 780 attttccata ca 792 2 1477 DNA Homosapiens misc_feature (1377)..(1377) Any nucleotide 2 gaattcccccaacagagcca agctctccat ctagtggaca gggaagctag cagcaaacct 60 tcccttcactacaaaacttc attgcttggc caaaaagaga gttaattcaa tgtagacatc 120 tatgtaggcaattaaaaacc tattgatgta taaaacagtt tgcattcatg gagggcaact 180 aaatacattctaggacttta taaaagatca ctttttattt atgcacaggg tggaacaaga 240 tggattatcaagtgtcaagt ccaatctatg acatcaatta ttatacatcg gagccctgcc 300 aaaaaatcaatgtgaagcaa atcgcagccc gcctcctgcc tccgctctac tcactggtgt 360 tcatctttggttttgtgggc aacatgctgg tcatcctcat cctgataaac tgcaaaaggc 420 tgaagagcatgactgacatc tacctgctca acctggccat ctctgacctg tttttccttc 480 ttactgtccccttctgggct cactatgctg ccgcccagtg ggactttgga aatacaatgt 540 gtcaactcttgacagggctc tattttatag gcttcttctc tggaatcttc ttcatcatcc 600 tcctgacaatcgataggtac ctggctgtcg tccatgctgt gtttgcttta aaagccagga 660 cggtcacctttggggtggtg acaagtgtga tcacttgggt ggtggctgtg tttgcgtctc 720 tcccaggaatcatctttacc agatctcaaa aagaaggtct tcattacacc tgcagctctc 780 attttccatacagtcagtat caattctgga agaatttcca gacattaaag atagtcatct 840 tggggctggtcctgccgctg cttgtcatgg tcatctgcta ctcgggaatc ctaaaaactc 900 tgcttcggtgtcgaaatgag aagaagaggc acagggctgt gaggcttatc ttcaccatca 960 tgattgtttattttctcttc tgggctccct acaacattgt ccttctcctg aacaccttcc 1020 aggaattctttggcctgaat aattgcagta gctctaacag gttggaccaa gctatgcagg 1080 tgacagagactcttgggatg acgcactgct gcatcaaccc catcatctat gcctttgtcg 1140 gggagaagttcagaaactac ctcttagtct tcttccaaaa gcacattgcc aaacgcttct 1200 gcaaatgctgttctattttc cagcaagagg ctcccgagcg agcaagctca gtttacaccc 1260 gatccactggggagcaggaa atatctgtgg gcttgtgaca cggactcaag tgggctggtg 1320 acccagtcagagttgtgcac atggcttagt tttcatacac agcctgggct gggggtnggt 1380 tggnngaggtcttttttaaa aggaagttac tgttatagag ggtctaagat tcatccattt 1440 atttggcatctgtttaaagt agattagatc cgaattc 1477 3 1442 DNA Homo sapiens 3 gaattcccccaacagagcca agctctccat ctagtggaca gggaagctag cagcaaacct 60 tcccttcactacaaaacttc attgcttggc caaaaagaga gttaattcaa tgtagacatc 120 tatgtaggcaattaaaaacc tattgatgta taaaacagtt tgcattcatg gagggcaact 180 aaatacattctaggacttta taaaagatca ctttttattt atgcacaggg tggaacaaga 240 tggattatcaagtgtcaagt ccaatctatg acatcaatta ttatacatcg gagccctgcc 300 aaaaaatcaatgtgaagcaa atcgcagccc gcctcctgcc tccgctctac tcactggtgt 360 tcatctttggttttgtgggc aacatgctgg tcatcctcat cctgataaac tgcaaaaggc 420 tgaagagcatgactgacatc tacctgctca acctggccat ctctgacctg tttttccttc 480 ttactgtccccttctgggct cactatgctg ccgcccagtg ggactttgga aatacaatgt 540 gtcaactcttgacagggctc tattttatag gcttcttctc tggaatcttc ttcatcatcc 600 tcctgacaatcgataggtac ctggctgtcg tccatgctgt gtttgcttta aaagccagga 660 cggtcacctttggggtggtg acaagtgtga tcacttgggt ggtggctgtg tttgcgtctc 720 tcccaggaatcatctttacc agatctcaaa aagaaggtct tcattacacc tgcagctctc 780 attttccatacattaaagat agtcatcttg gggctggtcc tgccgctgct tgtcatggtc 840 atctgctactcgggaatcct aaaaactctg cttcggtgtc gaaatgagaa gaagaggcac 900 agggctgtgaggcttatctt caccatcatg attgtttatt ttctcttctg ggctccctac 960 aacattgtccttctcctgaa caccttccag gaattctttg gcctgaataa ttgcagtagc 1020 tctaacaggttggaccaagc tatgcaggtg acagagactc ttgggatgac gcactgctgc 1080 atcaaccccatcatctatgc ctttgtcggg gagaagttca gaaactacct cttagtcttc 1140 ttccaaaagcacattgccaa acgcttctgc aaatgctgtt ctattttcca gcaagaggct 1200 cccgagcgagcaagctcagt ttacacccga tccactgggg agcaggaaat atctgtgggc 1260 ttgtgacacggactcaagtg ggctggtgac ccagtcagag ttgtgcacat ggcttagttt 1320 tcatacacagcctgggctgg gggtggttgg gaggtctttt ttaaaaggaa gttactgtta 1380 tagagggtctaagattcatc catttatttg gcatctgttt aaagtagatt agatccgaat 1440 tc 1442 4184 PRT Homo sapiens 4 Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr Asp IleAsn Tyr Tyr Thr 1 5 10 15 Ser Glu Pro Cys Gln Lys Ile Asn Val Lys GlnIle Ala Ala Arg Leu 20 25 30 Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile PheGly Phe Val Gly Asn 35 40 45 Met Leu Val Ile Leu Ile Leu Ile Asn Cys LysArg Leu Lys Ser Met 50 55 60 Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile SerAsp Leu Phe Phe Leu 65 70 75 80 Leu Thr Val Pro Phe Trp Ala His Tyr AlaAla Ala Gln Trp Asp Phe 85 90 95 Gly Asn Thr Met Cys Gln Leu Leu Thr GlyLeu Tyr Phe Ile Gly Phe 100 105 110 Phe Ser Gly Ile Phe Phe Ile Ile LeuLeu Thr Ile Asp Arg Tyr Leu 115 120 125 Ala Val Val His Ala Val Phe AlaLeu Lys Ala Arg Thr Val Thr Phe 130 135 140 Gly Val Val Thr Ser Val IleThr Trp Val Val Ala Val Phe Ala Ser 145 150 155 160 Leu Pro Gly Ile IlePhe Thr Arg Ser Gln Lys Glu Gly Leu His Tyr 165 170 175 Thr Cys Ser SerHis Phe Pro Tyr 180 5 352 PRT Homo sapiens 5 Met Asp Tyr Gln Val Ser SerPro Ile Tyr Asp Ile Asn Tyr Tyr Thr 1 5 10 15 Ser Glu Pro Cys Gln LysIle Asn Val Lys Gln Ile Ala Ala Arg Leu 20 25 30 Leu Pro Pro Leu Tyr SerLeu Val Phe Ile Phe Gly Phe Val Gly Asn 35 40 45 Met Leu Val Ile Leu IleLeu Ile Asn Cys Lys Arg Leu Lys Ser Met 50 55 60 Thr Asp Ile Tyr Leu LeuAsn Leu Ala Ile Ser Asp Leu Phe Phe Leu 65 70 75 80 Leu Thr Val Pro PheTrp Ala His Tyr Ala Ala Ala Gln Trp Asp Phe 85 90 95 Gly Asn Thr Met CysGln Leu Leu Thr Gly Leu Tyr Phe Ile Gly Phe 100 105 110 Phe Ser Gly IlePhe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu 115 120 125 Ala Val ValHis Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe 130 135 140 Gly ValVal Thr Ser Val Ile Thr Trp Val Val Ala Val Phe Ala Ser 145 150 155 160Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Lys Glu Gly Leu His Tyr 165 170175 Thr Cys Ser Ser His Phe Pro Tyr Ser Gln Tyr Gln Phe Trp Lys Asn 180185 190 Phe Gln Thr Leu Lys Ile Val Ile Leu Gly Leu Val Leu Pro Leu Leu195 200 205 Val Met Val Ile Cys Tyr Ser Gly Ile Leu Lys Thr Leu Leu ArgCys 210 215 220 Arg Asn Glu Lys Lys Arg His Arg Ala Val Arg Leu Ile PheThr Ile 225 230 235 240 Met Ile Val Tyr Phe Leu Phe Trp Ala Pro Tyr AsnIle Val Leu Leu 245 250 255 Leu Asn Thr Phe Gln Glu Phe Phe Gly Leu AsnAsn Cys Ser Ser Ser 260 265 270 Asn Arg Leu Asp Gln Ala Met Gln Val ThrGlu Thr Leu Gly Met Thr 275 280 285 His Cys Cys Ile Asn Pro Ile Ile TyrAla Phe Val Gly Glu Lys Phe 290 295 300 Arg Asn Tyr Leu Leu Val Phe PheGln Lys His Ile Ala Lys Arg Phe 305 310 315 320 Cys Lys Cys Cys Ser IlePhe Gln Gln Glu Ala Pro Glu Arg Ala Ser 325 330 335 Ser Val Tyr Thr ArgSer Thr Gly Glu Gln Glu Ile Ser Val Gly Leu 340 345 350 6 215 PRT Homosapiens 6 Met Asp Tyr Gln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr TyrThr 1 5 10 15 Ser Glu Pro Cys Gln Lys Ile Asn Val Lys Gln Ile Ala AlaArg Leu 20 25 30 Leu Pro Pro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe ValGly Asn 35 40 45 Met Leu Val Ile Leu Ile Leu Ile Asn Cys Lys Arg Leu LysSer Met 50 55 60 Thr Asp Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu PhePhe Leu 65 70 75 80 Leu Thr Val Pro Phe Trp Ala His Tyr Ala Ala Ala GlnTrp Asp Phe 85 90 95 Gly Asn Thr Met Cys Gln Leu Leu Thr Gly Leu Tyr PheIle Gly Phe 100 105 110 Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Thr IleAsp Arg Tyr Leu 115 120 125 Ala Val Val His Ala Val Phe Ala Leu Lys AlaArg Thr Val Thr Phe 130 135 140 Gly Val Val Thr Ser Val Ile Thr Trp ValVal Ala Val Phe Ala Ser 145 150 155 160 Leu Pro Gly Ile Ile Phe Thr ArgSer Gln Lys Glu Gly Leu His Tyr 165 170 175 Thr Cys Ser Ser His Phe ProTyr Ile Lys Asp Ser His Leu Gly Ala 180 185 190 Gly Pro Ala Ala Ala CysHis Gly His Leu Leu Leu Gly Asn Pro Lys 195 200 205 Asn Ser Ala Ser ValSer Lys 210 215 7 360 PRT Homo sapiens MISC_FEATURE (325)..(327) Xaa =any amino acid 7 Met Leu Ser Thr Ser Arg Ser Arg Phe Ile Arg Asn Thr AsnGlu Ser 1 5 10 15 Gly Glu Glu Val Thr Thr Phe Phe Asp Tyr Asp Tyr GlyAla Pro Cys 20 25 30 His Lys Phe Asp Val Lys Gln Ile Gly Ala Gln Leu LeuPro Pro Leu 35 40 45 Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn MetLeu Val Val 50 55 60 Leu Ile Leu Ile Asn Cys Lys Lys Leu Lys Cys Leu ThrAsp Ile Tyr 65 70 75 80 Leu Leu Asn Leu Ala Ile Ser Asp Leu Leu Phe IleIle Thr Leu Pro 85 90 95 Leu Trp Ala His Ser Ala Ala Asn Glu Trp Val PheGly Asn Ala Met 100 105 110 Cys Lys Leu Phe Thr Gly Leu Tyr His Ile GlyTyr Phe Gly Gly Ile 115 120 125 Phe Phe Ile Ile Leu Leu Thr Ile Asp ArgTyr Leu Ala Ile Val His 130 135 140 Ala Val Phe Ala Leu Lys Ala Arg ThrVal Thr Phe Gly Val Val Thr 145 150 155 160 Ser Val Ile Thr Trp Leu ValAla Val Phe Ala Ser Val Pro Gly Ile 165 170 175 Ile Phe Thr Lys Cys GlnLys Glu Asp Ser Val Tyr Val Cys Gly Pro 180 185 190 Tyr Phe Pro Arg GlyTrp Asn Asn Phe His Thr Ile Met Arg Asn Ile 195 200 205 Leu Gly Leu ValLeu Pro Leu Leu Ile Met Val Ile Cys Tyr Ser Gly 210 215 220 Ile Leu LysThr Leu Leu Arg Cys Arg Asn Glu Lys Lys Arg His Arg 225 230 235 240 AlaVal Arg Val Ile Phe Thr Ile Met Ile Val Tyr Phe Leu Phe Trp 245 250 255Thr Pro Tyr Asn Ile Val Ile Leu Leu Asn Thr Phe Gln Glu Phe Phe 260 265270 Gly Leu Ser Asn Cys Glu Ser Thr Ser Gln Leu Asp Gln Ala Ile Gln 275280 285 Val Thr Glu Thr Leu Gly Met Thr His Cys Cys Ile Asn Pro Ile Ile290 295 300 Tyr Ala Phe Val Gly Glu Lys Phe Arg Arg Tyr Ile Ser Val PhePhe 305 310 315 320 Arg Lys His Ile Xaa Xaa Xaa Phe Cys Lys Gln Cys ProVal Phe Tyr 325 330 335 Arg Glu Thr Val Asp Gly Val Thr Ser Thr Asn ThrPro Ser Thr Gly 340 345 350 Glu Gln Glu Val Ser Ala Gly Leu 355 360 8355 PRT Homo sapiens MISC_FEATURE (231)..(233) Xaa = amy amino acid 8Met Thr Thr Ser Ile Asp Thr Val Glu Thr Phe Gly Thr Thr Ser Tyr 1 5 1015 Tyr Asp Asp Val Gly Leu Leu Cys Glu Lys Ala Asp Thr Arg Ala Leu 20 2530 Met Ala Gln Phe Val Pro Pro Leu Tyr Ser Leu Val Phe Thr Val Gly 35 4045 Leu Ile Gly Asn Val Val Val Val Met Ile Leu Ile Lys Tyr Arg Arg 50 5560 Ile Arg Ile Met Thr Asn Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp 65 7075 80 Leu Leu Phe Ile Val Thr Leu Pro Phe Trp Thr His Tyr Val Arg Gly 8590 95 His Asn Trp Val Phe Gly His Gly Met Cys Asn Leu Ile Ser Gly Phe100 105 110 Tyr His Thr Gly Leu Tyr Ser Glu Ile Phe Phe Ile Ile Leu LeuThr 115 120 125 Ile Asp Arg Tyr Leu Ala Ile Val His Ala Val Phe Ala IleArg Ala 130 135 140 Arg Thr Val Thr Phe Gly Val Ile Thr Ser Ile Val ThrTrp Gly Ile 145 150 155 160 Ala Val Ile Ala Ala Leu Pro Glu Phe Ile PheTyr Glu Thr Glu Glu 165 170 175 Leu Phe Glu Glu Thr Ile Cys Ser Ala LeuTyr Pro Glu Asp Thr Val 180 185 190 Tyr Ser Trp Arg His Phe His Thr IleArg Met Thr Ile Phe Cys Leu 195 200 205 Val Leu Pro Leu Leu Val Met AlaIle Cys Tyr Thr Gly Ile Ile Lys 210 215 220 Thr Leu Leu Arg Cys Pro XaaXaa Xaa Lys Tyr Lys Ala Ile Arg Leu 225 230 235 240 Ile Phe Val Ile MetAla Val Phe Phe Ile Glu Trp Thr Pro Tyr Asn 245 250 255 Val Ala Ile LeuIle Ser Ser Tyr Gln Ser Leu Leu Phe Gly Asn Asn 260 265 270 Cys Glu ArgSer Lys His Leu Asp Leu Val Met Ile Val Thr Glu Val 275 280 285 Ile AlaTyr Ser His Cys Cys Met Asn Glu Val Ile Tyr Ala Phe Val 290 295 300 GlyGlu Arg Phe Arg Lys Tyr Ile Arg His Phe Phe His Arg His Leu 305 310 315320 Leu Met His Leu Gly Arg Tyr Ile Pro Phe Leu Pro Xaa Xaa Xaa Ile 325330 335 Glu Arg Ile Ser Ser Val Ser Pro Ser Thr Ala Glu Pro Glu Ile Ser340 345 350 Ile Val Phe 355 9 355 PRT Homo sapiens 9 Met Glu Thr Pro AsnThr Thr Glu Asp Tyr Asp Thr Thr Thr Glu Phe 1 5 10 15 Asp Tyr Gly AspAla Thr Pro Cys Gln Lys Val Asn Glu Arg Ala Phe 20 25 30 Gly Ala Gln LeuLeu Pro Pro Leu Tyr Ser Leu Val Phe Val Ile Gly 35 40 45 Leu Val Gly AsnIle Leu Val Val Leu Val Leu Val Gln Tyr Lys Arg 50 55 60 Leu Lys Asn MetThr Ser Ile Tyr Leu Leu Asn Leu Ala Ile Ser Asp 65 70 75 80 Leu Leu PheIle Phe Thr Leu Pro Phe Trp Ile Asp Tyr Lys Leu Lys 85 90 95 Asp Asp TrpVal Phe Gly Asp Ala Met Cys Lys Ile Ile Ser Gly Phe 100 105 110 Tyr TyrThr Gly Leu Tyr Ser Glu Ile Phe Phe Ile Ile Leu Leu Thr 115 120 125 IleAsp Arg Tyr Leu Ala Ile Val His Ala Val Phe Ala Ile Arg Ala 130 135 140Arg Thr Val Thr Phe Gly Val Ile Thr Ser Ile Ile Ile Trp Ala Ile 145 150155 160 Ala Ile Ile Ala Ser Met Pro Gly Leu Tyr Phe Ser Lys Thr Gln Trp165 170 175 Glu Phe Thr His His Thr Cys Ser Leu His Phe Pro His Glu SerLeu 180 185 190 Arg Glu Trp Lys Leu Phe Gln Ala Leu Lys Leu Asn Leu PheGly Leu 195 200 205 Val Leu Pro Leu Leu Val Met Ile Ile Cys Tyr Ile GlyIle Ile Lys 210 215 220 Ile Leu Leu Arg Arg Pro Asn Glu Lys Lys Ser LysAla Val Arg Leu 225 230 235 240 Ile Phe Val Ile Met Ile Ile Phe Phe LeuPhe Trp Ile Pro Tyr Asn 245 250 255 Leu Thr Ile Ile Ile Ser Val Phe GlnAsp Phe Leu Phe Thr His Glu 260 265 270 Cys Glu Gln Ser Arg His Leu AspLeu Ala Val Gln Val Thr Glu Val 275 280 285 Ile Ala Tyr Thr His Cys CysVal Asn Glu Val Ile Tyr Ala Phe Val 290 295 300 Gly Glu Arg Phe Arg LysTyr Ile Arg Gln Leu Glu His Arg Arg Val 305 310 315 320 Ala Val His LeuVal Lys Trp Leu Pro Phe Leu Ser Val Asp Arg Ile 325 330 335 Glu Arg ValSer Ser Thr Ser Pro Ser Thr Gly Glu His Glu Ile Ser 340 345 350 Ala GlyPhe 355 10 360 PRT Homo sapiens MISC_FEATURE (145)..(147) Xaa = anyamino acid 10 Met Asn Pro Thr Asp Ile Ala Asp Thr Thr Leu Asp Glu SerIle Tyr 1 5 10 15 Ser Asn Tyr Tyr Leu Tyr Glu Ser Ile Pro Lys Pro CysThr Lys Glu 20 25 30 Gly Ile Lys Ala Phe Gly Glu Leu Phe Leu Pro Pro LeuTyr Ser Leu 35 40 45 Val Glu Val Phe Gly Leu Ile Gly Asn Ser Val Val ValLeu Val Leu 50 55 60 Phe Lys Tyr Lys Arg Ile Arg Ser Met Thr Asp Val TyrLeu Leu Asn 65 70 75 80 Leu Ala Ile Ser Asp Leu Leu Phe Val Phe Ser LeuPro Phe Trp Gly 85 90 95 Tyr Tyr Ala Ala Asp Gln Trp Val Phe Gly Leu GlyIle Cys Lys Met 100 105 110 Ile Ser Trp Met Tyr Leu Val Gly Phe Tyr SerGly Ile Phe Phe Val 115 120 125 Met Ile Met Ser Ile Asp Arg Tyr Leu AlaIle Val His Ala Val Glu 130 135 140 Xaa Xaa Xaa Ala Arg Thr Ile Ile TyrGly Val Ile Thr Ser Leu Ala 145 150 155 160 Thr Trp Ser Val Ala Val PheAla Ser Leu Pro Gly Phe Ile Phe Ser 165 170 175 Thr Cys Tyr Thr Glu ArgAsn His Thr Tyr Cys Lys Thr Lys Tyr Ser 180 185 190 Leu Asn Ser Thr ThrTrp Lys Val Leu Ser Ser Leu Glu Ile Asn Ile 195 200 205 Leu Gly Leu ValIle Pro Leu Gly Ile Met Leu Phe Cys Tyr Ser Met 210 215 220 Ile Ile ArgThr Leu Gln His Cys Lys Asn Glu Lys Lys Asn Lys Ala 225 230 235 240 ValLys Met Ile Phe Ala Val Val Val Leu Phe Leu Gly Phe Trp Thr 245 250 255Pro Tyr Asn Ile Val Leu Phe Leu Glu Thr Leu Val Glu Leu Glu Val 260 265270 Ile Gln Asp Cys Thr Phe Glu Arg Tyr Leu Asp Tyr Ala Ile Gln Ala 275280 285 Thr Glu Thr Leu Ala Phe Val His Cys Cys Leu Asn Pro Ile Ile Tyr290 295 300 Phe Phe Leu Gly Glu Lys Phe Arg Lys Tyr Ile Ile Gln Leu PheLys 305 310 315 320 Xaa Xaa Xaa Gly Leu Phe Val Ile Cys Gln Tyr Cys GlyLeu Leu Gln 325 330 335 Ile Tyr Ser Ala Asp Thr Pro Ser Ser Ser Tyr ThrGln Ser Thr Met 340 345 350 Asp His Asp Leu His Asp Ala Leu 355 360 1149 PRT Homo sapiens 11 Phe Pro Tyr Ser Gln Tyr Gln Phe Trp Lys Asn PheGln Thr Leu Lys 1 5 10 15 Ile Val Ile Leu Gly Leu Val Leu Pro Leu LeuVal Met Val Ile Cys 20 25 30 Tyr Ser Gly Ile Leu Lys Thr Leu Leu Arg CysArg Asn Glu Lys Lys 35 40 45 Arg 12 147 DNA Homo sapiens 12 tttccatacagtcagtatca attctggaag aatttccaga cattaaagat agtcatcttg 60 gggctggtcctgccgctgct tgtcatggtc atctgctact cgggaatcct aaaaactctg 120 cttcggtgtcgaaatgagaa gaagagg 147 13 34 PRT Homo sapiens 13 Phe Pro Tyr Ile Lys AspSer His Leu Gly Ala Gly Pro Ala Ala Ala 1 5 10 15 Cys His Gly His LeuLeu Leu Gly Asn Pro Lys Asn Ser Ala Ser Val 20 25 30 Ser Lys 14 27 DNAArtificial Sequence primer_bind (1)..(27) Primer used to amplify thefull size coding region of the CCR5 ge ne 14 tcgaggatcc aagatggattatcaagt 27 15 27 DNA Artificial Sequence primer_bind (1)..(27) Primer toamplify the full size coding region of the CCR5 gene 15 ctgatctagagccatgtgca caactct 27 16 20 DNA Artificial Sequence primer_bind(1)..(20) Primer used to amplify CCR5 from genomic DNA samples 16cctggctgtc gtccatgctg 20 17 27 DNA Artificial Sequence primer_bind(1)..(27) primer used to amplify CCR5 from genomic DNA samples 17ctgatctaga gccatgtgca caactct 27 18 215 PRT Homo sapiens 18 Met Asp TyrGln Val Ser Ser Pro Ile Tyr Asp Ile Asn Tyr Tyr Thr 1 5 10 15 Ser GluPro Cys Gln Lys Ile Asn Val Lys Gln Ile Ala Ala Arg Leu 20 25 30 Leu ProPro Leu Tyr Ser Leu Val Phe Ile Phe Gly Phe Val Gly Asn 35 40 45 Met LeuVal Ile Leu Ile Leu Ile Asn Cys Lys Arg Leu Lys Ser Met 50 55 60 Thr AspIle Tyr Leu Leu Asn Leu Ala Ile Ser Asp Leu Phe Phe Leu 65 70 75 80 LeuThr Val Pro Phe Trp Ala His Tyr Ala Ala Ala Gln Trp Asp Phe 85 90 95 GlyAsn Thr Met Cys Gln Leu Leu Thr Gly Leu Tyr Phe Ile Gly Phe 100 105 110Phe Ser Gly Ile Phe Phe Ile Ile Leu Leu Thr Ile Asp Arg Tyr Leu 115 120125 Ala Val Val His Ala Val Phe Ala Leu Lys Ala Arg Thr Val Thr Phe 130135 140 Gly Val Val Thr Ser Val Ile Thr Trp Val Val Ala Val Phe Ala Ser145 150 155 160 Leu Pro Gly Ile Ile Phe Thr Arg Ser Gln Lys Glu Gly LeuHis Tyr 165 170 175 Thr Cys Ser Ser His Phe Pro Tyr Ile Lys Asp Ser HisLeu Gly Ala 180 185 190 Gly Pro Ala Ala Ala Cys His Gly His Leu Leu LeuGly Asn Pro Lys 195 200 205 Asn Ser Ala Ser Val Ser Lys 210 215

1. A method for identifying an organism comprising one or more cellswhich are resistant to HIV infection comprising: (a) obtaining abiological sample from said organism, wherein said sample comprises oneor more cells which may or may not comprise a nucleic acid moleculeencoding a polypeptide comprising SEQ ID NO: 6; (b) contacting saidbiological sample with an antibody which binds to a polypeptidecomprising amino acid residues 264 to 294 of SEQ ID NO: 6; and (c)determining if said antibody binds to said polypeptide present in saidbiological sample, wherein binding of said antibody to said polypeptideidentifies said organism as comprising one or more cells which areresistant to HIV infection.
 2. A method for identifying an organismcomprising one or more cells which are resistant to HIV infectioncomprising: (a) obtaining a biological sample from said organism,wherein said sample comprises one or more cells which may or may notcomprise a polypeptide encoded by a nucleic acid comprising residues 792to 884 of SEQ ID NO: 3; (b) contacting said biological sample with anantibody which binds to a polypeptide encoded by a nucleic acidcomprising residues 792 to 884 of SEQ ID NO: 3, wherein said polypeptidehas the same reading frame as the polypeptide of SEQ ID NO: 6; and (c)determining if said antibody binds to said polypeptide encoded by anucleic acid comprising residues 792 to 884 of SEQ ID NO: 3 present insaid biological sample, wherein binding of said antibody to saidpolypeptide identifies said organism as comprising one or more cellswhich are resistant to HIV infection.
 3. The method of claim 1 or 2,wherein said antibody is a monoclonal antibody.
 4. The method of claim 1or 2 wherein said HIV is HIV-1 or HIV-2.
 5. A method for identifying anorganism comprising one or more cells which are resistant to HIVinfection comprising: (a) obtaining a nucleic acid sample from saidorganism; (b) amplifying a portion of the sequence of SEQ ID NO: 3comprising residues 790-823 so as to generate an amplified product; and(c) detecting the presence of said amplified product, wherein thedetection of said amplified product identifies said organism ascomprising one or more cells which are resistant to HIV infection.
 6. Amethod for identifying an organism comprising one or more cells whichare resistant to HIV infection comprising: (a) obtaining a nucleic acidsample from said organism; (b) contacting said nucleic acid sample witha first and a second oligonucleotide primer, wherein said firstoligonucleotide primer is capable of specifically hybridizing with aportion of the sequence of SEQ ID NO: 3 comprising residues 790-792, andsaid second oligonucleotide primer specifically hybridizes with asequence comprised by the complement of the sequence of SEQ ID NO: 3,wherein the primer extension product of one oligonucleotide primer, whenseparated from its complement, can serve as a template for the synthesisof the extension product of the other primer; (c) subjecting theresulting mixture from step (b) to amplification comprising at least twocycles of nucleic acid strand separation, oligonucleotide primerannealing, and polymerase extension of annealed primers, so as togenerate an amplified product; and (d) detecting the presence of saidamplified product, wherein the detection of said amplified productidentifies said organism as comprising one or more cells which areresistant to HIV infection.
 7. The method of claim 5 or 6 wherein saidHIV is HIV-1 or HIV-2.
 8. A method for identifying an organismcomprising one or more cells which are resistant to HIV infectioncomprising: (a) obtaining a nucleic acid sample from said organism; (b)contacting said nucleic acid sample with a first and a secondoligonucleotide primer, wherein said first oligonucleotide primer iscapable of specifically hybridizing to a sequence within the portion ofSEQ ID NO: 2 comprising residues 791-823, and said secondoligonucleotide primer specifically hybridizes with a sequence comprisedby the complement of the sequence of SEQ ID NO: 2, wherein the primerextension product of one oligonucleotide primer, when separated from itscomplement, can serve as a template for the synthesis of the extensionproduct of the other primer; (c) subjecting the resulting mixture fromstep (b) to amplification comprising at least two cycles of nucleic acidstrand separation, oligonucleotide primer annealing, and polymeraseextension of annealed primers, so as to generate an amplified product ifresidues 791-823 of SEQ ID NO: 2 are present in the nucleic acid sample;and (d) detecting the presence or absence of said amplified product,wherein the absence of said amplified product identifies said organismas comprising one or more cells which are resistant to HIV infection. 9.A method for identifying an organism comprising one or more cells whichare resistant to HIV infection comprising: (a) obtaining a nucleic acidsample from said organism; (b) contacting said nucleic acid sample witha first and a second oligonucleotide primer, wherein said firstoligonucleotide primer is capable of specifically hybridizing with aportion of the sequence of SEQ ID NO: 2 upstream from residues 791-823,and said second oligonucleotide primer specifically hybridizes with asequence comprised by the complement of the sequence of SEQ ID NO: 2downstream from residues 791-823, wherein the primer extension productof one oligonucleotide primer, when separated from its complement, canserve as a template for the synthesis of the extension product of theother primer; (c) subjecting the resulting mixture from step (b) toamplification comprising at least two cycles of nucleic acid strandseparation, oligonucleotide primer annealing, and polymerase extensionof annealed primers, so as to generate an first amplified product; and(d) determining the size of the first amplification product obtained instep (c), wherein the size of said first amplification product isindicative of said organism comprising one or more cells which areresistant to HIV infection.
 10. The method of claim 9, wherein said stepof determining comprises comparing the size of said amplificationproduct to the actual or predicted size of a second amplificationproduct obtained using said primers of step (b) in an amplificationreaction wherein a nucleic acid molecule having the sequence of SEQ IDNO: 2 is uses as a template nucleic acid, wherein if the size of saidfirst amplification product is smaller than the size of said secondamplification product, then said organism is identified as comprisingone or more cells which are resistant to HIV infection.